In this four year two phase (R2l/R33) project we will develop and apply new approaches for obtaining broad, sensitive, and quantitative measurements of protein abundances in human tumors. The overall approach aims to advance the study of cancer proteomes by more rapidly identifying many more proteins and more precisely measuring their relative abundances from much smaller samples than currently feasible with existing methodologies. Our approach will utilize proteome-wide stable isotope and biotin labeling of both cysteine-containing polypeptides and phosphopeptides to obtain broad proteome coverage. Th,~s will be combined with a single step very high resolution capillary liquid chromatography (LC) separation and ultra-high sensitivity and high mass accuracy of measurements using new Fourier transform ion cyclotron resonance (FTICR) mass spectrometry instrumentation developed at our laboratory. The approach will be at least 3 orders of magnitude more sensitive than existing 2-D P AGE methodologies and able to rapidly identify and measure relative expression levels for thousands of proteins in a single analysis. Phase 1 of this project will integrate and provide an initial demonstration of (a) the protein stable-isotope labeling and sample processing methodologies, (b) the ability to conduct high resolution capillary LC separations with "data-directed" FTICR tandem mass spectrometry (MS/MS), and (c) application of these methods for protein identifications and precise quantitation of small breast tumor samples. The technological approach will exploit very accurate mass measurements and multiplexed-MS/MS methods for protein identification, and the abundances of stable-isotope cysteine-containing polypeptides and phosphopeptides from whole-proteome tryptic digestions to a obtain precision of approximately 10% uncertainty. The technology will be further advanced and refined in Phase 2 to allow ultra-sensitive proteome-wide precision profiling of proteins from small numbers of cells from breast tissues ( e.g. obtained by micro-dissection) will be evaluated. Specifically, the Phase 2 application will involve the pilot application of the technology to globally determine relative protein abundances and phosphorylation states of proteomes relevant to breast cancer progression, including identifying distinctive protein profiles of ductal carcinoma in situ, tumor and normal breast epithelium.